Zn-T8 is a novel autoantigen in type 1 diabetes (T1D) which is encoded by a polymorphic SLC30A8 gene. The role of Zn-T8 as a target in T cell-mediated islet autoimmunity has remained largely unexplored. To our knowledge, there are no reports on the Zn-T8-specific T cell responses in the preclinical phase of diabetes progression. We propose to determine the role of Zn-T8-specific T cell responses as a novel biomarker in the prediction of T1D by investigating longitudinal changes in these responses in pre-diabetic subjects and in T1D families. Our hypothesis is that CD4+ T cell responses to Zn-T8 provide an early predictive marker for progression to T1D, and that appearance of Zn-T8-specific T cells may precede the emergence of Zn-T8 autoantibodies. The relationship and hierarchy between Zn-T8-specific T cell responses and other biochemical autoantibodies (insulin, GAD65, and IA-2) will also be evaluated. We also aim to assess the phenotype/genotype differences of the Zn-T8 specific responses. In the family study the comparison between autoantibody positive and negative siblings and the T1D child is expected to reveal a potential regulatory phenotype which could discriminate the subjects who are unlikely to progress to disease despite of the shared genetic background. We predict that there will be differences in the Zn-T8 epitope utilization and/or T cell reactivity when subjects are stratified by SLC30A8 polymorphism. Finally we will test the hypothesis that polymorphism in the SLC30A8 gene has an effect on the T cell responses to proinsulin through altered proinsulin processing, which has been reported to be associated with this genomic variant. This is a novel genotype-phenotype analysis, in which the Zn-T8 genotype will be compared with the CD4 T cell profile. In our preliminary study we demonstrated a high prevalence of Zn-T8 specific CD4+ T cells in the preclinical phase in the children who progressed to T1D. Most prediabetic subjects displayed T cell responses to multiple Zn-T8 epitopes suggesting that determinant spreading may play a role in the pathogenesis of T1D and may be used as a predictive tool to monitor progression of the disease prior to the clinical onset. The overall goal of the project is to provide a novel approach to T cel-mediated autoimmunity in T1D which could be applied to prediction.